PROJECT SUMMARY High throughput screens (HTS) are routinely performed in both drug discovery and toxicity testing, where libraries of compounds are screened for their effects on a given biological endpoint (i.e. druggable target, cell toxicity, etc.). A challenge with this approach is that drugs/chemicals act by targeting specific biomolecules, but are often metabolized to different compounds before they reach their target in the body. Since most HTS reporter cell lines exhibit limited drug metabolism, they often fail to report drugs or chemicals that are metabolized to a more active form (resulting in a false negative) or are rapidly detoxified/cleared (resulting in a false positive). Enhancing existing reporter assays with metabolic competence to increase the in vivo predictability of these assays while retaining their compatibility with existing HTS infrastructure would be invaluable in identifying novel drug candidates and identifying potentially hazardous chemicals. We have developed a microtiter plate based device for high-throughput co-culture dubbed the Metabolism Integrated Co-culture RepOrter - MicroTiter plate or MICRO-MT (Fig. 1). The MICRO-MT incorporates physiological levels of chemical metabolism into HTS assays by enabling the co-culture of metabolically competent liver cells with existing reporter cell assays in a technically simple manner that seamlessly integrates with existing robotic infrastructure (e.g. no new equipment required). While co-culture of a reporter cell assay with hepatocytes has been pursued as a viable solution to this problem, traditional Transwell co-culture assays are not well suited for robotic HTS due to increased robotic handling and analytical incompatibility. The MICRO-MT is a simple HTS retrofit that fits seamlessly into existing infrastructure. Preliminary data with the fungal natural product, aflatoxin B1 (AFB1) demonstrated that Hek293 cells in monoculture were insensitive to AFB1, but that HepaRG co-culture with Hek293 cells in the MICRO-MT conferred assay sensitivity of Hek293 cells to AFB1. Comprehensive characterization of the MICRO-MT will move the assay toward commercial use in the HTStox market, drug development, metabolite discovery and beyond.